Mineralization and alkaline phosphatase activity in collagen lattices populated by human osteoblasts

Adult human osteoblastic cells were grown in a native type I collagen gel. Proliferation and viability analyses showed that cells rapidly stopped divi ding and became blocked in the G0G1 phase (91% on day 13). Carboxyfluoresce in diacetate cell staining and flow cytometry showed that osteoblasts were viable for the first 16 days and then viability decreased (58% viable cells on day 22). Osteoblasts were able to retract the matrix. Betaglycerophosph ate (beta GP) stimulated the deposition of mineral particles in the collage n network, and electron probe microanalysis showed that they were principal ly calcium and phosphorus, with a Ca/P ratio of about 1.7. Various times of beta GP supply were tested. We compared 10 mM beta GP added only once at d ay 0, or continuously from day 0, day 8, or day 21. Mineralization was obse rved in conditions where beta GP was added at day 0. Furthermore, 10 mM bet a GP added once during gel preparation was sufficient to induce mineralizat ion with mineral accumulation up to day 15 whereas the speed of the gel con traction decreased. In every condition, cultures expressed high alkaline ph osphatase (ALP) levels as early as day 3, which decreased afterwards. These kinetics might explain why the other conditions did not prove favorable to the mineralization process. The model was used to study the influence of b locking gel retraction. Blocking retraction delayed the ALP activity decrea se, but had no effect on mineralization. In conclusion, human adult osteobl asts cultured in native collagen gel stopped proliferation and underwent mi neralization very early. This model should be used to investigate the influ ence of effecters on the early stages of culture.